From 2010.igem.org

TEAM ALBERTA

Optimizations

Project Timeline: Click on an image to see more information

PCR Using Universal Primers

Aug 27, 2010:

Tested different amount of dNTPs added to 30uL PCR reactions.

Results: Diluting the dNTPs to a final concentration of 0.2mM yielded cleaner PCR products compared to dilutions of higher dNTP concentrations.

Aug 31, 2010:

Experimented with the dNTP:MgCl₂ ratios to see if it yielded cleaner and/or more PCR products while increasing the reaction volume to 50uL to increase the accuracy of measuring reagents.

Results: Diluting MgCl₂ to a final concentration of 2mM led to more specific PCR production without sacrificing too much of the quantity of desired products.

Sept 1, 2010:

Tested different annealing temperatures ranging from 55^oC to 70^oC.

Results: We did not find significant variations between the different annealing temperatures. We decided to go with 62^oC since it yielded more products.

Sept 2, 2010:

Tested different cycles of 15, 20 and 25.

Results: 25 Cycles produced a large enough quantity of PCR products for the amount of time required to run the program compared to the other cycles, as long with 30 Cycles. Depending on the amount of PCR products needed, 25 or 30 cycles will be used.

Sept 10, 2010:

Tested how the modification of using 1ng of template to 10ng will affect PCR efficiency.

Results: No significance difference was seen; therefore, using 1ng of template will be sufficient for PCR reactions.

BsaI Digestions

Sept 7, 2010:

Test different amounts of DNA samples – 0.5ug, 1ug, 1.5ug and 2ug – that can be digested by BsaI at 37^oC and 50^oC for one hour with and without BSA.

Results: At 1.5 ug and up, the digestion is not cut to completion at 37^oC. All samples are cut at 50^oC without BSA.

Sept 8, 2010:

Test how many ug of DNA could be digested in one hour with only 1uL (10 units) of BsaI at 50^oC. We are using samples containing 2.5ug, 5ug and 7.5ug of DNA.

Results: While 2.5ug is completely cut, at 5ug, the DNA is not completely cut. However, majority of the plasmid is cut when comparing band intensities of the undigested to digested bands.

Sept 10, 2010:

Test 2 to 4 hour digestion incubations at 37^oC and 50^oC with BsaI to see whether enzyme activity is maintained longer at 37^oC.

Results: BsaI enzymatic activity is maintained longer at 37^oC while at 50^oC BsaI enzymatic activity is lost in 2 hours. However, there were no complete digestions by BsaI at either temperature.

Sept 13, 2010:

Test how long it will take to digest 5ug of DNA with 1uL BsaI at 50^oC.

Results: Loss of enzymatic activity in one hour.

Test to see if 10ug of DNA will be digested overnight at 37^oC (approximately 16 hours).

Sept 14, 2010:

Results: The DNA plasmid is not cut to completion.

Team:Alberta/Notebook/Optimizations